Potential antidepressants: Pharmacology of 2-(4-methyl piperazin-1-yl)-1,8- naphthyridine-3-carbonitrile in rodent behavioural models

Article abstract of DOI:10.1691/ph.2007.12.7552

Serotonin type 3 (5-HT₃) antagonists, which find an unflinching place in the management of nausea and emesis are presently screened for their neuro-pharmacological potential in various animal models. In the present study, 2-(4-methyl piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile (NA-2) with an optimal log P and pA₂ value comparable to that of ondansetron was screened in rodent models of depression. The acute and chronic (14 days) treatment of the synthetic compound exhibited antidepressant-like effects at the lower dose levels in mice forced swim test (FST). A typical and similar dose-immobility profile was observed in both mice FST and tail suspension test (TST). Interaction studies in FST revealed the reversal of mCPP induced immobility, attenuation of antidepressant effects of fluoxetine and desipramine. Chronic NA-2 treatment restored the behavioural deficits in olfactory bulbectomized (OBX) rats as indicated by reduction in hyperactivity in novel open field test. This preliminary study points to a serotonergic mechanism behind the antidepressant-like effects of NA-2 and invigorates further investigation of analogous compounds in various other models of depression.

Full text of DOI:10.1691/ph.2007.12.7552

ORIGINAL ARTICLES
Pharmacy Group, Birla Institute of Technology & Science, Pilani, Rajasthan, India
Potential antidepressants: Pharmacology of 2-(4-methyl piperazin-1-yl)-1,8-
naphthyridine-3-carbonitrile in rodent behavioural models
R. Mahesh, R. Rajkumar, B. Minasri, R. Venkatesha Perumal
Received February 24, 2007, accepted April 30, 2007
Ramamoorthy Rajkumar, Research Scholar, Pharmacy Group, Birla Institute of Technology & Science,
Pilani-333 031, Rajasthan, India
rajkumarrna@yahoo.com
Pharmazie 62: 919–924 (2007)
doi: 10.1691/ph.2007.12.7552
Serotonin type 3 (5-HT3) antagonists, which find an unflinching place in the management of nausea
and emesis are presently screened for their neuro-pharmacological potential in various animal models.
In the present study, 2-(4-methyl piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile (NA-2) with an optimal
log P and pA2 value comparable to that of ondansetron was screened in rodent models of depression.
The acute and chronic (14 days) treatment of the synthetic compound exhibited antidepressant-like
effects at the lower dose levels in mice forced swim test (FST). A typical and similar dose-immobility
profile was observed in both mice FST and tail suspension test (TST). Interaction studies in FST
revealed the reversal of mCPP induced immobility, attenuation of antidepressant effects of fluoxetine
and desipramine. Chronic NA-2 treatment restored the behavioural deficits in olfactory bulbectomized
(OBX) rats as indicated by reduction in hyperactivity in novel open field test. This preliminary study
points to a serotonergic mechanism behind the antidepressant-like effects of NA-2 and invigorates
further investigation of analogous compounds in various other models of depression.
1. Introduction
piperazines were designed and synthesized. Microwave
assisted synthesis of 2-(4-substitued piperazin-1-yl)-1,8-
naphthyridine-3-carbonitriles was carried out giving a
series of 12 compounds which were characterized by
spectral (IR, 1H NMR and mass) and elemental analy-
sis. The compounds were tested for their 5-HT3 antago-
nistic property in isolated guinea pig ileum and the pA2
values were determined against 2-methyl-5 hydroxytrypt-
amine (Mahesh et al. 2004). Anticipating a dual use in
managing both vomiting and depression associated with
cancer, we investigated the antidepressant potential of
5-HT3 antagonists.
Forced swim test (Porsolt et al. 1977; Bourin et al. 2002)
and tail suspension test (Steru et al. 1985; Thierry et al.
1986) are the most extensively used mice behavioural
models for antidepressant screening. More than 25 years
of immense research has strengthened the validities of
these models, positioning it vitally among the various
other models in whole animal antidepressant test battery
(Bourin et al. 2001, 2005). Bilateral olfactory bulb abla-
tion in rats induces various behavioural deficits as a result
of secondary structural and functional neural alterations
(Song and Leonard 2005; Cairncross et al. 1979) and anti-
depressants are proved to restore the normal behaviour
(Van Reizen and Leonard 1990). The hyperactivity exhib-
ited by olfactory bulbectomised rats in a novel open field
arena is reversed by repeated administration of conven-
tional antidepressants. The olfactory bulbectomy (OBX)
model has also revealed the antidepressant-like effects of
agents affecting the 5-HT receptor subtypes (Kelly et al.
1997).
Serotonin type-3 receptor antagonists (5-HT3 RAs) are
currently promising in the management of nausea and vo-
miting associated with cancer chemotherapy (Jantunen
et al. 1997; Mahesh et al. 2005). Interestingly, in the last
decade, these molecules have been extensively screened
for their neuro-psychopharmacological potentials in var-
ious pre-clinical and few clinical studies (Israili 2001;
Wolf 2000). The involvement of 5-HT3 receptors in the
neural pathways of depression has prompted research,
considering these receptors as possible antidepressant drug
targets (Costall and Naylor 2004). However, the complete
neurobiological aspects behind depression are still antici-
pated and inconsistent results have been obtained with 5-
HT3 RAs (Greenshaw 1993). These receptors seem to play
a partial yet notable role in depression (Bhatnagar et al.
2004; Redrobe and Bourin 1997). Depression affects 15%
of the population worldwide (Hirschfeld et al. 1997) and
is associated with both cancer and cancer chemotherapy
(Pirl 2004; Massie 2004). Nevertheless, no special treat-
ment modes have been devised or adopted for the man-
agement of cancer related depression, incidence of which
is three times more than the depressive disorder itself
(Fisch 2004).
Supported by the three component pharmacophore model
(Hibert et al. 1990) of 5-HT3 RAs, several series of com-
pounds have been designed and screened for 5-HT3 antag-
onism. Taking into account the cost involved in synthesis
of 5-HT3 RAs (compounds with pharmacophore design
suggested by Hibert et al.) with chiral centers heteroaryl
Pharmazie 62 (2007) 12
919

ORIGINAL ARTICLES
Table 1: Data showing the pA2 and log P values of 2-(4-sub-
350
300
250
200
150
100
50
stituted piperazin-1-yl)-1,8-naphthyridine-3-carboni-
trile series of compounds (NA series)
*
CN
N
N
N
0
Vehicle 0.005 0.010 0.100 1.000 10.000 PAR mCPP
N
(15 mg/kg)
(1mg/kg)
NA-2 (µg/kg)
R
Acute treatment
Chronik treatment
Compd.
R
pA2
log P
Fig. 1: Influence of NA-2, PAR or mCPP treatment on spontaneous loco-
motor activity. Results are expressed as mean Locomotor scores
recorded in a period of 10 minutes. Error bars represent S.E.M.
NA-1
NA-2
NA-3
NA-4
NA-5
NA-6
NA-7
NA-8
–H
–CH3
–C2H5
–CH2–CH=CH2
–C6H5
–CH2-C6H5
o-OCH3–C6H4
m-OCH3–C6H4
p-OCH3–C6H4
p-NO2–C6H4
p-Cl–C6H4
6.5
1.39
1.84
2.27
1.68
2.35
2.38
2.44
2.46
2.53
1.53
2.97
1.91
2.58
*
7.4
7.1
8.2
5.7
5.1
5.4
6.0
4.9
3.8
*
p < 0.05 compared to corresponding vehicle treated group (n ¼ 6)
A
240
200
NA-9
*
NA-10
NA-11
NA-12
Ondansetron
160
<3.0
3.9
6.9
*
*
120
80
40
0
*
2-pyridyl
*
*
*
*
*
*
*
2-(4-methyl piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile, screened for antidepres-
sant potential
0.005
Vehicle
0.010 0.100 1.000 10.000 PAR
(15 mg/kg)
NA-2 (µg/kg)
Acute treatment Chronic treatment
In the present study, 2-(4-methyl piperazin-1-yl)-1,8-
naphthyridine-3-carbonitrile (NA-2) which exhibited an
optimal log P and pA2 value comparable to ondansetron
(Mahesh et al. 2004) was selected for preliminary antide-
pressant screening in the standard rodent models of de-
pression viz. forced swim and tail suspension test in mice
and open field behaviour of olfactory bulbectomised
(OBX) rats. Interaction studies with fluoxetine (FLX), de-
sipramine (DMI) and meta-chlorophenyl piperazine
(mCPP) were carried out in mice FST.
B
*
60
50
40
30
20
10
0
*
*
*
*
*
*
*
2. Investigations and results
Vehicle 0.005 0.010 0.100 1.000 10.000 PAR
(15 mg/kg)
The log P values of a series of 12 compounds are depicted
in Table 1. NA-2 possessed log P and pA2 value of 1.84
and 7.4 respectively.
ΝΑ-2 (µg/kg)
Acute treatment
Chronic treatment
Fig. 2: A. Effects of acute and chronic treatment of NA-2 and PAR on
duration of immobility in mice FST.Results are expressed as mean
2.1. Spontaneous locomotor activity
*
duration of immobility. Error bars represent S.E.M. p < 0.05
compared to corresponding vehicle treated group (n ¼ 12)
Acute and chronic treatment of NA-2 and paroxetine
(PAR) did not influence the locomotion at tested dose le-
vels. However, chronic NA-2 (1 and 10 mg/kg) treatment
decreased the locomotor activity of mice compared to ve-
hicle treatment (Fig. 1), however the results were statisti-
cally insignificant. Chronic mCPP (1 mg/kg) treatment de-
creased the locomotion significantly as compared to
vehicle control.
B. Effects of acute and chronic treatment of NA-2 and PAR on
swimming behaviour in Mice FST.Results are expressed as mean
number of quadrants crossed. Error bars represent S.E.M.
*
p < 0.05 compared to corresponding vehicle treated group
(n ¼ 12)
300
250
*
*
*
*
200
150
100
*
*
2.2. Dose response study in FST and TST
*
*
*
Acute and chronic treatment with NA-2 showed significant
(p < 0.05) anti-immobility effects in both the models of
depression with peak activity at 0.01 mg/kg, but the effects
were lesser compared to PAR (15 mg/kg). In the FST,
NA-2 exhibited a decreased anti-immobility effect and de-
creased swimming episodes with an increase in dose.
Chronic treatment with NA-2 slightly decreased the dura-
tion of immobility (Fig. 2A) and increased the swimming
episodes (Fig. 2B) as compared to the acute treatment.
NA-2 treatment exhibited a similar dose-immobility pat-
tern in TST (Fig. 3).
50
0
Vehicle 0.005 0.010 0.100 1.000 10.000 PAR
(15 mg/kg)
NA-2 (µg/kg)
Chronic treatment
Aute treatment
Fig. 3: Effects of acute and chronic treatment of NA-2 and PAR on dura-
tion of immobility in Mice TST. Results are expressed as mean
*
duration of immobility. Error bars represent S.E.M. p < 0.05.
Compared to corresponding vehicle treated group (n ¼ 10)
920
Pharmazie 62 (2007) 12

ORIGINAL ARTICLES
Table 2: Effect of NA-2 pre-treatment on mCPP, FLX and DMI modulated depressive states in mice FST
Group
(n)
Treatment
Duration of Immobility (Sec)
Swimming Episodes
(Number of quadrants crossed)
1 (12)
2 (12)
3 (12)
4 (12)
5 (12)
6 (12)
7 (12)
8 (12)
9 (12)
10 (8)
11 (8)
12 (8)
Vehicle (Distilled water)
NA-2 (0.01 mg/kg)
NA-2 (0.01 mg/kg for 14 days )
mCPP (1 mg/kg)
FLX (10 mg/kg)
168.17 ꢀ 3.14
11.33 ꢀ 1.03
22.25 ꢀ 1.62a, c
24.75 ꢀ 1.75a, c
7.25 ꢀ 0.59
95.25 ꢀ 3.40a, c
93.12 ꢀ 4.15a, c
197.08 ꢀ 3.09a
72.33 ꢀ 1.82a, b
101.17 ꢀ 2.57a
169.00 ꢀ 4.13b, c
118.75 ꢀ 3.90a, b, d
123.67 ꢀ 2.39a, b, e
165.25 ꢀ 3.25b, c
89.38 ꢀ 5.46a
33.08 ꢀ 1.54a, b
20.33 ꢀ 1.04a
8.50 ꢀ 0.82
DMI (10 mg/kg)
NA-2 (0.01 mg/kg) þ mCPP (1 mg/kg)
NA-2 (0.01 mg/kg) þ FLX (10 mg/kg)
NA-2 (0.01 mg/kg) þ DMI (10 mg/kg)
NA-2 (0.01 mg/kg for 14 days) þ mCPP (1 mg/kg)
NA-2 (0.01 mg/kg for 14 days) þ FLX (10 mg/kg)
NA-2 (0.01 mg/kg for 14 days) þ DMI (10 mg/kg)
22.75 ꢀ 1.01a, d
16.17 ꢀ 0.68a, b
12.50 ꢀ 0.74b, c
29.50 ꢀ 3.35a
17.13 ꢀ 1.87
135.88 ꢀ 4.78a, e
a
b
c
d
Values represent mean ꢀ S.E.M. p < 0.05 compared to vehicle group, p < 0.05 compared to NA-2 treated group, p < 0.05 compared to mCPP treated group. p < 0.05
e
compared to FLX treated group. p < 0.05 compared to DMI treated group
2.3. Interaction study
FST, TST and pilot study. Chronic (14 days) treatment
with NA-2 showed statistically significant (p < 0.05) re-
duction in ambulation (depicted as horizontal activity in
Fig. 4), rearing (depicted as vertical activity in Fig. 5) and
defecation (Fig. 6) in OBX rats compared to the vehicle
treated group. NA-2 (0.1 mg/kg) showed antidepressant-
like effects and PAR was the most effective among all
treatments. NA-2 (0.1 mg/kg) also showed statistically sig-
nificant increase in the time spent in grooming, scratching
and licking (Fig. 7). None of the treatment showed any
statistically significant change in the behaviour of sham
operated animals.
A single dose of NA-2 (0.01 mg/kg) showed a significant
(p < 0.05) decrease in the duration of immobility and a
significant (p < 0.05) increase in swimming episodes. The
antidepressant-like effects of NA-2 (acute and chronic)
were weaker than that of fluoxetine (FLX 10 mg/kg) and
desipramine (DMI 10 mg/kg). Acute pretreatment of NA-2
significantly (p < 0.05) attenuated the antidepressant ef-
fects of FLX, whereas both acute and chronic pretreatment
significantly (p < 0.05) reduced the antidepressant effect
of DMI. The mCPP induced immobility was reversed by
acute and chronic pretreatment with NA-2 (0.01 mg/kg).
Results are depicted in Table 2.
3. Discussion
2.4. Olfactory bulbectomy induced hyperactivity
Alkyl substitutions at the 4th position of the piperazine, in-
creased the log P value whereas allyl substitution reduced
it. Increase in hydrophobicity was most prominent with
aromatic substitutions. A log P around 2 is considered op-
timum for a centrally (blood brain barrier permeable) act-
The lower dose levels of NA-2 (0.01 and 0.1 mg/kg) were
selected for screening in the olfactory bulbectomy model
of depression in rats based on the data obtained from the
Horizontal activity
160
Vertical activity
30
140
25
*
120
*
20
100
*
*
80
60
40
*
15
10
5
0
20
0
OBX
Sham
OBX
Sham
Fig. 4: Effect of NA-2 and PAR on the horizontal activity of OBX and
sham rats in the open field test. All drug/vehicle administrations
were done once a day for 14 days. Results are expressed as mean
Fig. 5: Effect of NA-2 and PAR on the vertical activity of OBX and sham
rats in the open field test. All drug/vehicle administrations were
done once a day for 14 days. Results are expressed as mean num-
*
*
number of squares crossed. Error bars represent S.E.M; p < 0.05
ber of rearing episodes. Error bars represent S.E.M. p < 0.05
compared to vehicle treated OBX group (n ¼ 6)
compared to vehicle treated OBX group (n ¼ 6)
OBX þ vehicle
OBX þ NA ꢁ 2 ð0:01 mg=kgÞ
OBX þ vehicle
OBX þ NA ꢁ 2 ð0:01 mg=kgÞ
OBX þ NA ꢁ 2 ð0:1 mg=kgÞ
OBX þ PAR ð10 mg=kgÞ
Sham þ PAR ð10 mg=kgÞ
OBX þ NA ꢁ 2 ð0:1 mg=kgÞ
OBX þ PAR ð10 mg=kgÞ
Sham þ PAR ð10 mg=kgÞ
Sham þ vehicle
Sham þ NA ꢁ 2 ð0:01 mg=kgÞ
Sham þ vehicle
Sham þ NA ꢁ 2 ð0:01 mg=kgÞ
Sham þ NA ꢁ 2 ð0:1 mg=kgÞ
Sham þ NA ꢁ 2 ð0:1 mg=kgÞ
Pharmazie 62 (2007) 12
921

ORIGINAL ARTICLES
effects as indicated by the spontaneous locomotor activity
Defecation
test. The characteristic dose-immobility profile and partial
effects (at lower doses) on swimming behaviour needs fo-
cus. The 5-HT3 receptor is claimed as one of the substrates
for behavioural effects of antidepressants and for definitive
evaluation of antidepressant potential of any 5-HT3 RAs,
interaction study with selective serotonin reuptake inhibi-
tors (SSRIs), is essential (Cryan et al. 2005). Ondansetron
pretreatment (single dose) was found to enhance antide-
pressant effects of FLX and it has been demonstrated in
mice FST that antagonism of 5-HT3 receptors may have a
partial role in anti-immobility effect of SSRIs and antag-
onism of 5-HT2A/C is vital for antidepressant effects of tri-
cyclic antidepressants (Redrobe and Bourin 1997).
9
8
7
6
5
4
*
*
3
2
*
1
0
In the current study, acute pre-treatment of NA-2 signifi-
cantly attenuated the anti-depressant effects of FLX and
DMI. mCPP, a non selective 5-HT2 agonist induces immo-
bility in rat FST possibly mediated by 5-HT2C or 5-HT1B
receptors (Cryan and Lucki 2000). Acute mCPP (1 mg/kg)
treatment increased the duration of immobility and de-
creased the swimming behaviour but did not affect sponta-
neous locomotor activity and hence was used in the inter-
action study. However, chronic treatment showed
significant decrease in locomotor scores. Pretreatment
(chronic slightly effective than acute) with NA-2 (0.01 mg/
kg) reversed mCPP(acute) induced immobility indicating
the probable serotonergic mechanism involving 5-HT2 and
5-HT1B receptors, behind the antidepressant-like effect of
NA-2.
OBX
Sham
Fig. 6: Effect of NA-2 and PAR on defecation of OBX and Sham rats in
the open field test. All drug/vehicle administrations were done once
a day for 14 days. Results are expressed as mean number of fecal
*
pellets. Error bars represent S.E.M. p < 0.05 compared to vehicle
treated OBX group (n ¼ 6)
OBX þ vehicle
OBX þ NA ꢁ 2 ð0:01 mg=kgÞ
OBX þ NA ꢁ 2 ð0:1 mg=kgÞ
OBX þ PAR ð10 mg=kgÞ
Sham þ PAR ð10 mg=kgÞ
Sham þ vehicle
Sham þ NA ꢁ 2 ð0:01 mg=kgÞ
Sham þ NA ꢁ 2 ð0:1 mg=kgÞ
ing, orally bioactive compound (Ter Laak et al. 1994).
NA-2, which possessed log P and pA2 values of 1.84 and
7.4 respectively, was hence found suitable for antidepres-
sant assays.
The rat olfactory bulbectomy model of chronic depression
(with adequate face and predictive validity) is known to
espial novel agents that are unmarked by more conven-
tional models of depression (Bourin et al. 2001). The
OBX rats exhibited a specific abnormal behavioural pat-
tern in the brightly lit, circular, open field arena (Song and
Leonard 2005; Kelly and Leonard 1994). The various be-
havioural changes included increased horizontal and verti-
cal activity, increased defecation and decreased grooming
compared to sham controls (Slotkin et al. 1999). Such
changes are reversed by antidepressants of many pharma-
cological classes (Van Riezen and Leonard 1990), and in
the present study PAR (10 mg/kg) significantly (p < 0.05)
reversed the behavioural changes. Olfactory bulbectomy
was proposed as a model for agitated hyposerotonergic de-
pression since the antidepressant-responsive changes in lo-
comotor activity in bulbectomised rats are associated with
reduction in serotonin neurotransmission (Lumia et al.
1992). In addition, 5-HT2 receptor density is increased in
OBX rats, which are normalized by chronic antidepressant
treatment (Earley et al. 1994). NA-2 at both doses (0.1 mg/
kg being more effective) reduced the hyperactivity in bul-
bectomised rats but the restoration of behavioural deficits
was weaker than that of PAR.
Acute and chronic treatment of NA-2 showed antidepres-
sant-like effect with similar dose-immobility profiles in
both models of depression. Assessment of anti-depressant
potential is interfered by possible hyper-locomotive prop-
erty of a test substance leading to false positive results
(Porsolt et al. 1978). The antidepressant-like effects of
NA-2 in the FST and TST are not due to hyper-locomotive
Grooming, scratching and licking episodes
25
*
*
20
15
10
5
#
Based on this behavioural study, though viewed as a 5-
HT3 RA, it is speculative to attribute 5-HT2 receptor invol-
vement in the antidepressant-like effect of NA-2 indicated
by the reversal of both mCPP induced immobility in mice
FST and behavioural anomalies in OBX rats. However,
studies probing the affinity of NA-2 on 5-HT1B, 5-HT2, 5-
HT3 receptor subtypes and serotonin transporters are re-
quired to confirm its precise mechanism of action.
To conclude, the results from three reliable antidepressant
behavioural models indicate that 2-(4-methylpiperazin-1-
yl)-1,8-naphthyridine-3-carbonitrile, exhibits antidepres-
sant-like effects at the dose levels mentioned. The study
also encourages analogues molecules for antidepressant
screening. Though no abnormal behaviour was observed
0
OBX
Sham
Fig. 7: Effect of NA-2 and PAR on the time spent by OBX and sham rats
in grooming, scratching and licking in the open field test. All drug/
vehicle administrations were done once a day for 14 days. Results
are expressed as mean of time spent in Grooming, scratching and
*
licking. Error bars represent S.E.M. p < 0.05 compared to vehicle
treated OBX group. #p < 0.05 compared to vehicle treated sham
group (n ¼ 6)
OBX þ vehicle
OBX þ NA ꢁ 2 ð0:01 mg=kgÞ
OBX þ NA ꢁ 2 ð0:1 mg=kgÞ
OBX þ PAR ð10 mg=kgÞ
Sham þ PAR ð10 mg=kgÞ
Sham þ vehicle
Sham þ NA ꢁ 2 ð0:01 mg=kgÞ
Sham þ NA ꢁ 2 ð0:1 mg=kgÞ
922
Pharmazie 62 (2007) 12

ORIGINAL ARTICLES
30 cm, diameter: 22.5 cm) containing a depth of 15 cm of water main-
at the tested dose levels, assessment of the safety profile
of the molecule is essential.
tained at 23–25 ꢂC and were left in the water for 6 min. A mouse was
judged immobile if it floated in the water in an upright position and exhib-
ited only small movements to keep its head above the water or made other
passive movements. The duration of immobility was recorded during the
last 4 min of the 6 min test. The swimming episodes were recorded as the
number of quadrants (demarcated at the base of the cylinder) crossed.
4. Experimental
4.1. Animals
Experiments on animals were approved by the Institutional Animal Ethics
Committee of Birla Institute of Technology and Science, Pilani, India (Pro-
tocol No.IAEC/RES/4/1, dated 22. 09. 04; IAEC/RES/7/1, dated
24. 04. 06). Albino mice of either sex (18–25 g) and male Wistar rats
(250–300 g) were obtained from Hissar Agricultural University, Hissar,
Haryana, India and maintained in standard laboratory conditions with food
(standard pellet chow feed) and filtered water ad libitum. The animals were
used only once for each experiment.
4.4.3. Tail suspension test
The method mentioned elsewhere (Steru et al. 1985, Rodrigues et al. 2002)
with slight modifications was adopted. To mention in brief, mice were in-
dividually suspended by the tail to a horizontal bar (distance from floor
was 50 cm) using scotch tape (distance from tip of tail was approximately
1 cm). Typically, mice demonstrated several escape-oriented behaviors in-
terspersed with temporally increasing bouts of immobility. The 6-min test
session was manually observed. The parameter recorded was the number
of seconds spent immobile.
4.2. Compound administration
Fluoxetine (FLX) and paroxetine (PAR) were obtained from Sun Pharma-
ceuticals, and Ipca Laboratories, India respectively. Desipramine (DMI)
was obtained from Sigma Chemicals and meta-chlorophenyl piperazine
(mCPP) was obtained from Lancaster chemicals, USA. Chloral hydrate
was obtained from Reidel (India) Chemicals Pvt. Ltd. The hydrochloride
salt of NA-2 (Mahesh et al. 2004) or drugs were solublised in sterile dis-
tilled water and were freshly prepared for use.
In the acute study, groups were treated individually with a single dose of
NA-2 (0.005, 0.01, 0.1, 1, 10 mg/kg). All administrations were made intra-
peritoneally. Thirty minutes after treatment, the mice were subjected to lo-
comotor or antidepressant screening. In the chronic study, NA-2 (0.005,
0.01, 0.1, 1, 10 mg/kg) was administered orally, once a day for 14 days. On
the 14th day, 1 h after the last dose the animals were subjected to locomotor
or antidepressant assays. For interaction studies NA-2 and antidepressants/
mCPP were administered intraperitoneally, 45 and 30 min respectively be-
fore testing as per the procedure mentioned elsewhere (Redrobe and Bourin
1997; Bourin et al. 2002). The acute and chronic dose response profiles of
NA-2 were studied in both FST and TST. Interaction studies of NA-2 with
conventional antidepressants/mCPP were carried out in the FST.
In OBX rat model of depression, NA-2 (0.01 and 0.1 mg/kg), vehicle or
PAR (10 mg/kg) was administered once a day for 14 days to OBX rats,
after a post-surgical rehabilitation period of 21 days. The OBX rats were
then subjected to open field test, 20 h after the last dose to avoid the possi-
ble acute effects of the drug treatment. The doses of standard antidepres-
sants were selected from the pilot studies conducted in our laboratory. All
administrations and testing were done between 10–14 h. To avoid bias, all
behavioural observations were carried out by trained experimenters, who
had no information on the treatment.
4.4.4. Olfactory bulbectomy
4.4.4.1. Surgery
Bilateral olfactory bulb ablation was performed in rats anesthetized with
chloral hydrate (400 mg/kg) as described elsewhere (Kelly et al. 1997; Van
Riezen and Leonard 1990), with slight modifications. The skull was ex-
posed by a midline incision and burr holes (2 mm in diameter) were drilled
8 mm anterior to bregma and 2 mm on either side of the midline at a point
corresponding to the posterior margin of the orbit of the eye. The olfactory
bulbs were removed by suction, the holes were then filled with haemostatic
sponge in order to control excessive bleeding and the scalp was sutured.
To prevent infection, the animals were given Sulprim injection (each ml
containing 200 and 40 mg of sulphadiazine and trimethoprim respectively),
intramuscularly (0.2 ml/300 g) once a day for 3 days, post-surgery. Sham-
operated animals received the same surgical treatment, but the bulbs were
left intact. The animals were given 21 days recovery period following the
surgery prior to antidepressant screening. All the animals were handled
daily by the experimentor throughout the recovery period to reduce aggres-
sive behavior (Leonard and Tuite 1981). Animals which exhibited any
other abnormal behaviour were excluded from the study. The drug treat-
ment was started on the 21st day after surgery.
4.4.4.2. Openfield behaviers
Olfactory bulbectomized (OBX) and sham control rats were subjected to
an open field test on the 14th day of chronic drug/vehicle administration.
The open field exploration was conducted as described by Kelly et al.
(1997) with slight modifications. The apparatus consisted of a circular (90-
cm diameter) arena with 75-cm high aluminum walls and floor equally
divided into 10 cm squares. A 60 W light bulb was positioned 90 cm
above the base of the arena, which was the only source of illumination in
the testing room. Each animal was individually placed in the center of the
open field apparatus and the following parameters were noted for 5 min by
2 trained observers. Ambulation scores (number of squares crossed) and
number of rearing episodes were noted as horizontal and vertical activity,
respectively. The time spent in grooming/scratching/licking and number of
fecal pellets were also measured. The apparatus was cleaned with ethyl
alcohol and dried between trials.
4.3. Chemistry
Na-2 was synthesized according to Mahesh et al. (2004). Briefly, a mixture
of 2 aminonicotinaldehyde (Majewicz and Caluwe 1974), ethyl cyanoace-
tate and piperidine was triturated at room temperature for about 10 min.
The completion of reaction was confirmed by TLC. The solid thus ob-
tained was treated with water, filtered, and recrystallised from dimethyl for-
mamide (DMF)-water mixture to give 2-oxo-1,2-dihydro-1,8 naphthyri-
dine-3-carbonitrile. This was refluxed with phosphorus oxychloride for 1 h
in the presence of DMF, cooled to room temperature and treated with ice
water. The resulting solution was slowly basified with aqueous sodium hy-
droxide (40%) with cooling. The separated product was washed with water
and recrystallised to give 2-chloro-1,2-dihydro-1,8 naphthyridine-3-carboni-
trile. Microwave irradiation of this intermediate with 1-methyl piperazine
in the presence of potassium carbonate in DMF for 5 min gave 2-(4-methyl
piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile (NA-2). The compound
(m.r. 152–154 ꢂC) was characterized by Spectral (IR, NMR, and MS) and
elemental analysis. This free base was dissolved in chloroform and hydro-
chloride gas was purged to obtain the hydrochloride salt. The freely water
soluble compound was stored in a cool dry place until use.
4.5. Statistical analysis
The locomotor scores, duration of immobility, swimming episodes and the
behavioural scores in the open field test were expressed as mean ꢀ SEM.
The data was subjected to one-way ANOVA followed by post hoc dun-
nett’s ‘T3’ test and the level of statistical significance was fixed at
p < 0.05.
Acknowledgements: We thank Sun Pharmaceutical Industries Ltd, Ipca La-
boratories, Mumbai, India for providing fluoxetine hydrochloride and par-
oxetine hydrochloride, respectively, as gift samples in a very short notice.
4.4. Behavioural screening
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